The acute chest syndrome (ACS) is a leading cause of death among sickle cell disease (SCD) patients. Currently, the mechanism of lung injury in ACS remains largely unknown and no preventative treatments exist. SCD patients hospitalized with vaso-occlusive pain crisis often develop ACS within the ensuing days, suggesting a potential role for vaso-occlusion (i.e. blood vessel blockage) in ACS. We aim to elucidate the cellular and molecular mechanism behind pulmonary vaso-occlusion to enable the development of new molecular targeted treatments to prevent ACS. We have developed a multi-photon excitation (MPE) fluorescence microscopy based approach to visualize blood cell trafficking and injury within the lung of live transgenic SCD mice. SCD mice have constitutively active neutrophils, platelets and endothelial cells. To trigger vaso-occlusion and lung injury, SCD mice must experience a `second hit', such as an inflammatory stimulus. We used intravenous (IV) bacterial lipopolysaccharide (LPS) to induce experimental ACS in SCD mice, as LPS challenge is a well-established model of acute lung injury in mice and LPS has been found to cause vaso-occlusion in the cremaster venules of SCD mice. We found that IV LPS challenge in SCD mice induced occlusion of the pulmonary arteriole, which involved platelet capture by neutrophils, micro-thrombus formation and cessation of blood flow. These findings inform our hypothesis that ACS in SCD is initiated by in situ neutrophil-platelet micro-embolism of the pre-capillary pulmonary arterioles, which involves P-selectin-PSGL-1 and GPIb?-Mac-1 enabled platelet nucleation on arrested neutrophils. We will test this hypothesis with the following aims: 1) To assess whether IV LPS induces more potent and lethal acute lung injury in SCD versus control mice. 2) To test the hypothesis that IV LPS induced lung injury in SCD mice is preceded by neutrophil-platelet micro-embolism of the pre- capillary pulmonary arterioles. 3) To determine whether blocking neutrophil Mac-1 and platelet P-selectin prevents IV LPS induced pulmonary arteriole micro-embolism and subsequent lung injury in SCD mice. The candidate is currently a postdoctoral scholar in the Vascular Medicine Institute at the University of Pittsburgh. This proposal will provide Dr. Bennewitz with an in-depth training in basic science and the role of molecular interactions in SCD, and with a multitude of career development opportunities available through the training environment at the VMI. Mark Gladwin, MD, Prithu Sundd, PhD, and Solomon Ofori-Acquah, PhD, will provide Dr. Bennewitz with the clinical and basic science expertise and career guidance necessary to thrive as a young investigator. Through mentorship, scientific presentations, networking, group meetings, peer reviewed publications, career development opportunities, and the preparation of a K award, Dr. Bennewitz will receive the necessary training needed to transition into an independent investigator and become an expert in the field of cell trafficking.